Inhibition of nucleic acid polymerases by endonuclease v-cleavable oligonucleotide ligands

ABSTRACT

Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases, comprising cleaving the aptamer by endonuclease V enzymatic activity to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, thereby activating DNA synthesis activity of the DNA polymerase in a reaction mixture. Mixtures for use in methods of the invention are also provided. In some aspects, the oligonucleotide aptamer comprises one or more deoxyinosine nucleotides providing for aptamer-specific recognition and cleavage of the aptamer by the endonuclease V enzymatic activity. Exemplary oligonucleotide aptamers, mixtures and methods employing endonuclease V enzymatic activity are provided. The methods can be practiced using kits comprising a DNA polymerase-binding oligonucleotide aptamer and at least one endonuclease V enzymatic activity having oligonucleotide aptamer-specific recognition to provide for specific cleavage of the aptamer by the endonuclease V enzymatic activity.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.16/210,510, filed Dec. 5, 2018, which claims the benefit of U.S.Provisional Patent Application No. 62/595,451, filed Dec. 6, 2017, thedisclosures of which are herein incorporated by reference in theirentirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “0102384-001US1 SequenceListing.txt,” which was created on Dec. 5, 2017, and is 11 KB in size,are hereby incorporated by reference in their entirety.

BACKGROUND

Aspects of the present invention relate generally to improved methods ofblocking DNA polymerase activity with oligonucleotide aptamers at lowreaction temperatures, and restoring the enzyme activity upon raisingthe reaction temperature (e.g., hot-start methods).

DNA polymerases are enzymes used for synthesis of DNA strands by primerextension, wherein the polymerase-catalyzed DNA synthesis may beinitiated by oligonucleotide primers hybridized to a complementarytemplate DNA. Initiating DNA synthesis from this template-hybridizedprimer, DNA polymerases create complementary DNA strands in the presenceof corresponding nucleotide 5′-triphosphates. Sequence specificity ofnucleotide polymerization, when the oligonucleotide primers bindexclusively to the desired sites and nowhere else, is an importantrequirement in many applications wherein DNA synthesis is used. However,the efficiency and fidelity of DNA synthesis can be reduced when primershybridize to non-complementary DNAs, leading to synthesis of incorrectDNA sequences.

Many so-called ‘Hot Start’ methods have been developed to avoidincorrect primer extension products (e.g., see Paul, N., et al. (2010),for review). One of the most common techniques is based on use ofoligonucleotide aptamers (Jayasena, S. D., 1999). Aptamers offer anumber of advantages over other reported methods. Using a method ofmolecular evolution (SELEX), they can be quickly engineered in a testtube and then readily and inexpensively manufactured by chemicalsynthesis. Ideally, an aptamer should: (i) completely block DNApolymerase at low temperatures, and (ii) provide no blockage effect atthe desired elevated reaction temperature. Unfortunately, this is verydifficult, if not impossible to achieve, and the aptamer structureusually represents a compromise between these two key requirements. Newmethods, therefore, are needed to improve control of aptamer activity inreaction mixtures containing DNA polymerases.

Particular aspects provide methods of activating an aptamer-inactivatedDNA polymerase, comprising: providing a reaction mixture suitable forDNA synthesis, the reaction mixture comprising (i) a DNA polymerase,(ii) an endonuclease V-cleavable oligonucleotide aptamer that binds tothe DNA polymerase, wherein the oligonucleotide aptamer is present in anamount effective to inhibit DNA synthesis activity of the DNA polymerasein the reaction mixture, and (iii) an endonuclease V enzymatic activity;and cleaving the aptamer by the endonuclease V enzymatic activity toreduce or eliminate binding of the oligonucleotide aptamer to the DNApolymerase, thereby activating the DNA synthesis activity of the DNApolymerase, to increase DNA synthesis in the reaction mixture. In themethods, cleaving may be facilitated using a reaction temperature thatfacilitates both DNA polymerase activity and the endonuclease Venzymatic activity. In the methods, cleaving may be facilitated byincreasing the temperature of the reaction mixture from a firsttemperature to a second temperature that more strongly facilitates theendonuclease V enzymatic activity. In the methods, providing a reactionmixture suitable for DNA synthesis may comprise dissolving a dried formof at least one of said (i) DNA polymerase, (ii) endonucleaseV-cleavable oligonucleotide aptamer, and (iii) endonuclease V enzymaticactivity into an aqueous solution. DNA synthesis in the methods mayresult in DNA amplification in the reaction mixture (e.g., wherein theDNA amplification comprises PCR) DNA synthesis in the methods maycomprise an isothermal amplification reaction. The methods may comprisedetecting the presence of a target DNA and/or measuring an amount of atarget DNA in the reaction mixture. In the methods, the oligonucleotideaptamer may comprise one or more deoxyinosine nucleotides. In themethods, the oligonucleotide aptamer may have a stem-loop structure,wherein one or more deoxyinosine nucleotides may be incorporated intothe stem segment of the stem-loop structure, and/or wherein one or moredeoxyinosine nucleotides may be incorporated into the loop segment ofthe stem-loop structure. In the methods, the loop of the stem-loopstructure, may, for example, comprise a nucleotide sequence selectedfrom the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:22),5′-TTCTIAGCGTTT-3′ (SEQ ID NO:23), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:24),5′-TTCIIAGCGTTT-3′ (SEQ ID NO:25), 5′-TTCITAICGTTT-3′ (SEQ ID NO:26),5′-TTCTIAICGTTT-3′ (SEQ ID NO:27), and 5′-TTCIITAICGTTT-3′ (SEQ IDNO:28). In the methods, the loop of the stem-loop structure may, forexample, comprise one of the nucleotide sequences 5′-TTCITAGCGTTT-3′(SEQ ID NO:22), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:23) or 5′-TTCTTAICGTTT-3′(SEQ ID NO:24). In the methods, the endonuclease V enzymatic activitymay comprise, for example, Thermotoga maritima endonuclease V enzymaticactivity.

Additional aspects provide kits for activating an aptamer-inactivatedDNA polymerase, comprising: an endonuclease V enzymatic activity; and aDNA polymerase-binding oligonucleotide aptamer cleavable by anendonuclease V enzymatic activity. In the kits, the oligonucleotideaptamer may comprise one or more deoxyinosine nucleotides. In the kits,the oligonucleotide aptamer may comprise a stem-loop structure. In thekits, one or more deoxyinosine nucleotides may be located in the stemsegment of the stem-loop structure, and/or one or more deoxyinosinenucleotides may be located in the loop segment of the stem-loopstructure. In the kits, the loop of the stem-loop structure, may, forexample, comprise a nucleotide sequence selected from the groupconsisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCTIAGCGTTT-3′ (SEQID NO:23), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:24), 5′-TTCIIAGCGTTT-3′ (SEQ IDNO:25), 5′-TTCITAICGTTT-3′ (SEQ ID NO:26), 5′-TTCTIAICGTTT-3′ (SEQ IDNO:27), and 5′-TTCIITAICGTTT-3′ (SEQ ID NO:28). In the kits, the loop ofthe stem-loop structure may, for example, comprise one of the nucleotidesequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCTIAGCGTTT-3′ (SEQ IDNO:23) or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:24). In the kits, theendonuclease V enzymatic activity may comprise, for example, Thermotogamaritima endonuclease V enzymatic activity.

Further aspects provide reaction mixtures for use in a method of DNAsynthesis, which reaction mixture comprises: a DNA polymerase; anendonuclease V-cleavable oligonucleotide aptamer that binds reversiblyto the DNA polymerase, wherein the oligonucleotide aptamer is present inan amount effective to inhibit DNA synthesis activity of the DNApolymerase in the reaction mixture; and an endonuclease V enzymaticactivity capable of cleaving (or effective to cleave) theoligonucleotide aptamer to reduce or eliminate binding of theoligonucleotide aptamer to the DNA polymerase, thereby activating theDNA synthesis activity of the DNA polymerase. In the reaction mixtures,the DNA polymerase activity and/or the endonuclease V enzymatic activitymay be temperature-dependent. In the reaction mixtures, the endonucleaseV enzymatic activity may increase when the reaction mixture is heatedfrom a first temperature value to a second temperature value thatpromotes the endonuclease V enzymatic activity. In the reactionmixtures, the DNA polymerase, oligonucleotide aptamer, and endonucleaseV enzymatic activity may be in a dried state. In the reaction mixtures,the oligonucleotide aptamer may comprise one or more deoxyinosinenucleotides. In the reaction mixtures, the oligonucleotide aptamer maycomprise a stem-loop structure. In the reaction mixtures, one or moredeoxyinosine nucleotides may be located in the stem segment of thestem-loop structure, and/or one or more deoxyinosine nucleotides may belocated in the loop segment of the stem-loop structure. In the reactionmixtures, the loop of the stem-loop structure, may, for example,comprise a nucleotide sequence selected from the group consisting of5′-TTCITAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:23),5′-TTCTTAICGTTT-3′ (SEQ ID NO:24), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:25),5′-TTCITAICGTTT-3′ (SEQ ID NO:26), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:27),and 5′-TTCIITAICGTTT-3′ (SEQ ID NO:28). In the reaction mixtures, theloop of the stem-loop structure may, for example, comprise one of thenucleotide sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:22),5′-TTCTIAGCGTTT-3′ (SEQ ID NO:23) or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:24).In the reaction mixtures, the endonuclease V enzymatic activity maycomprise, for example, Thermotoga maritima endonuclease V enzymaticactivity. The reaction mixtures may further comprise one or more ofdATP, dCTP, dGTP, and/or dTTP, and/or Mg²⁺ ion.

Yet further aspects provide oligonucleotide aptamers, comprising anucleic acid sequence that forms a hairpin structure, having a stem anda loop portion, that binds to a DNA polymerase, wherein the stem and/orthe loop portion comprises one or more deoxyinosine nucleotides, andwherein the loop portion comprises a nucleotide sequence5′-TTCTTAGCGTTT-3′ (SEQ ID NO:21) that may be substituted withdeoxyinosine at one or more of positions 4, 5, and 7. In theoligonucleotide aptamers, the loop portion may comprise the nucleotidesequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:21). In the oligonucleotideaptamers, one or more deoxyinosine nucleotides may be located in thestem segment of the stem-loop structure, and/or one or more deoxyinosinenucleotides may be located in the loop segment of the stem-loopstructure. In the oligonucleotide aptamers, the loop of the stem-loopstructure, may, for example, comprise a nucleotide sequence selectedfrom the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:22),5′-TTCTIAGCGTTT-3′ (SEQ ID NO:23), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:24),5′-TTCIIAGCGTTT-3′ (SEQ ID NO:25), 5′-TTCITAICGTTT-3′ (SEQ ID NO:26),5′-TTCTIAICGTTT-3′ (SEQ ID NO:27), and 5′-TTCIITAICGTTT-3′ (SEQ IDNO:28). In the oligonucleotide aptamers, the loop of the stem-loopstructure may, for example, comprise one of the nucleotide sequences5′-TTCITAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:23) or5′-TTCTTAICGTTT-3′ (SEQ ID NO:24). In the oligonucleotide aptamers, theone or more deoxyinosine nucleotides may render the oligonucleotideaptamer cleavable by an endonuclease V enzymatic activity (e.g., whereinthe endonuclease V enzymatic activity may comprise Thermotoga maritimaendonuclease V enzymatic activity).

Still further aspects provide compositions comprising a DNA polymerasecomplexed with an oligonucleotide aptamer as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows, according to particular exemplary aspects, a portion of ahuman β2-microglobulin gene sequence (SEQ ID NO:4), forward and reverseprimers (SEQ ID NOS:1-2, respectively) and a 22-mer fluorescent probe(SEQ ID NO:3), which were used in exemplary 5′-nuclease PCR assays ofthe present invention from which exemplary results are shown in FIGS. 3,5, and 7. The primers and probe are shown aligned with an amplifiedβ2-microglobulin fragment sequence in 5′→3′ orientation as indicated.

FIG. 2 shows, according to particular exemplary aspects, structures ofstem-loop deoxyribonucleotide aptamers used in exemplary 5′-nuclease PCRassays of the present invention from which exemplary results are shownin FIGS. 3A and 3B. The symbol “I” indicates the presence ofdeoxyriboinosine nucleotides in aptamers SEQ ID NOS:6-10 that were usedin experiments with endonuclease V. Arrows point to the expectedendonuclease V cleavage positions.

FIGS. 3A and 3B show, according to particular exemplary aspects, theresults of fluorescence monitoring of reaction mixtures during PCR(real-time curves) in the presence of the individual aptamers listed inFIG. 2. Sequences of the amplified β2-microglobulin template, primersand 22-mer FRET probe used in these PCR assays are as shown in FIG. 1.Dashed lines are real-time curves obtained in the absence of anyaptamer. Experiments were conducted in the absence (FIG. 3A) or presence(FIG. 3B) of T. maritima endonuclease V. Experimental details areprovided below in “Example 2.”

FIG. 4 shows, according to particular exemplary aspects, structures ofadditional stem-loop deoxyribonucleotide aptamers used in the5′-nuclease PCR assays from which exemplary results are shown in FIGS.5A and 5B. Unmodified aptamer SEQ ID NO:11 has the same natural basecomposition as unmodified aptamer SEQ ID NO:5 of FIG. 2, but wasproduced by exchanging the upper and lower sequences of SEQ ID NO:5within the double-stranded stem portion delineated by the dashed box.This design approach provided further elucidation of the effect ofguanosine-to-inosine substitutions in the 5′ duplex sequence of aptamersSEQ ID NOS:12-16, which are modified versions of SEQ ID NO:11 (thesymbol “I” means a deoxyriboinosine nucleotide). Arrows point toendonuclease V cleavage positions.

FIGS. 5A and 5B show, according to particular exemplary aspects, theresults of fluorescence monitoring of reaction mixtures during PCR(real-time curves) in the presence of the individual aptamers listed inFIG. 4. Sequences of the amplified β2-microglobulin template, primersand 22-mer FRET probe used in the assays are as shown in FIG. 1. Dashedlines are real-time curves obtained in the absence of any aptamer.Experiments were conducted in the absence (FIG. 5A) or presence (FIG.5B) of T. maritima endonuclease V. Experimental details are providedherein below under “Example 2.”

FIG. 6 shows, according to particular exemplary aspects, structures offour additional stem-loop deoxyribonucleotide aptamers SEQ ID NOS:17-20that are derivatives of the aptamer SEQ ID NO:5 of FIG. 2 incorporatingdeoxyinosine nucleotide (symbol “I”) in the loop portion at variouspositions. These aptamers were used in the 5′-nuclease PCR assays ofFIGS. 7A and 7B. Arrows point to the anticipated endonuclease V cleavagepositions.

FIGS. 7A and 7B show, according to particular exemplary aspects, theresults of fluorescence monitoring of reaction mixtures during PCR(real-time curves) in the presence of the individual aptamers listed inFIG. 6. Sequences of the amplified β2-microglobulin template, primersand 22-mer FRET probe used in the assays are as shown in FIG. 1. Dashedlines are real-time curves obtained in the absence of the aptamers.Experiments were conducted in the absence (FIG. 7A) or presence (FIG.7B) of T. maritima endonuclease V. Experimental details are providedherein below under “Example 2.”

FIG. 8 shows, according to particular exemplary aspects, a reactionscheme used in the assays of FIGS. 9A through 9F to detect and measureDNA polymerase activity. The depicted hairpin-like fluorescent probe SEQID NO:29 was designed to have a G/C-rich stem in the duplex segment anda 5′- . . . GAA . . . hairpin-stabilizing loop to provide for use atelevated temperatures (e.g., up to 60-65° C.) (e.g., see Yoshizawa S.,et al, 1994). Extension of this hairpin-like probe in a reaction bufferin the presence of deoxyribonucleoside 5′-triphosphates (dNTPs) and aDNA polymerase results in a fluorescent signal that directly correlateswith the polymerase activity in the reaction.

FIGS. 9A through 9F show, according to particular exemplary aspects,endonuclease V-induced activation of Taq (FIG. 9D), Phusion® (FIG. 9B),Q5® (FIG. 9C), Vent® (FIG. 9A), Deep Vent® (FIG. 9E) and Bst largefragment (FIG. 9F) DNA polymerases that were initially deactivated(i.e., “inhibited” or “blocked”) by the presence of aptamer SEQ ID NO:6(♦ curves). FIGS. 9A through 9F also show the change of fluorescencewith time in the absence of endonuclease V for the aptamer-blocked (∘)and unblocked (□) DNA polymerase. The DNA polymerase activity wasmonitored by extension of the self-priming hairpin-like fluorescentprobe SEQ ID NO:29 (see FIG. 8), which was present in the reactionmixture with all four dNTPs in a magnesium-containing buffer. In allcases experiments were performed at 60 or 65° C., as indicated in eachfigure. The structure of aptamer SEQ ID NO:6 is shown in FIG. 2, anddetails of the experimental setup, results analysis and conclusions areprovided below in “Example 3.”

DETAILED DESCRIPTION Definitions

Terms and symbols of biochemistry, nucleic acid chemistry, molecularbiology and molecular genetics used herein follow those of standardtreatises and texts in the field (e.g., Sambrook, J., et al., 1989;Kornberg, A. and Baker, T., 1992; Gait, M. J., ed., 1984; Lehninger, A.L., 1975; Eckstein, F., ed., 1991, and the like). To facilitateunderstanding of particular exemplary aspects of the invention, a numberof terms are discussed below.

In particular aspects, “aptamer” or “oligonucleotide aptamer” refersherein to an oligonucleotide that is capable of binding to a DNApolymerase and blocking its DNA synthesis enzymatic activity. Theaptamers can be linear molecules and/or capable to form secondarystructures such as hairpin or stem-loop structures, etc. Examples ofaptamers and methods of selection (design) can be found, for instance,in Yakimovich, O. Yu., et al., (2003); Jayasena, S. D. (1999); U.S. Pat.No. 5,693,502 to Gold, L. and Jayasena, S. D., which are incorporatedhere by reference. The phrase “aptamer, that binds to the DNApolymerase, in an amount sufficient to inhibit DNA synthesis activity ofthe DNA polymerase,” as used herein, means that the DNA synthesisactivity of the DNA polymerase is at least partially inhibited (e.g.,inhibited to a level in the range of from about 1% to about 99.99%). Anylevel of aptamer inhibition of the DNA synthesis activity of the DNApolymerase can provide an advantage for DNA synthesis, and thusaccording to particular preferred hot start aspects of the presentinvention, the DNA synthesis activity of the DNA polymerase issubstantially inhibited (e.g., inhibited to a level in the range ofabout 80% to 99.99%, or to any subrange or level therein), or completely(100%) inhibited, providing an advantage over other ‘hot start’technologies (e.g., Paul, N., et al., 2010). Likewise, in the disclosedmethods, “cleaving the aptamer by the Endonuclease V enzymatic activityto reduce or eliminate the binding of the oligonucleotide aptamer to theDNA polymerase and activate the DNA synthesis activity of the DNApolymerase” is preferably complete (100%) or substantially complete(e.g., inhibited to a level in the range of about 80% to 99.99%, or toany subrange or level therein), but can be partial (e.g., inhibited to alevel in the range of from about 1% to about 99.99%), as exemplifiedherein (e.g., FIGS. 3, 5, 7, and 9).

An oligonucleotide aptamer may comprise ribo- or 2′-deoxyribonucleotidesor a combination thereof. Oligonucleotide aptamers may be modified.Regarding the aptamers herein, the terms “modified” and “modification”are used in two different aspects, wherein the aptamers can be (i)modified synthetically, e.g. during the oligonucleotide synthesis, and(ii) enzymatically-modified in the context of or during DNA synthesisreactions. Synthetically, the aptamers may incorporate any kind and/ornumber of structural modifications across the length of the aptamer(e.g., in the middle or at the ends of the oligonucleotide chain). Theterm “structural modifications” refers to any chemical substances suchas atoms, moieties, residues, polymers, linkers or nucleotide analogs,etc., which are usually of a synthetic nature and which are not commonlypresent in naturally-occurring nucleic acids. As used herein, the term“structural modifications” also include nucleoside or nucleotide analogswhich are rarely present in naturally-occurring nucleic acids includingbut not limited to inosine, 5-bromouracil, 5-methylcytosine,5-iodouracil, 2-aminoadenosine, 6-methyladenosine, pseudouridine,deoxyuridine, and the like. In particular embodiments, the aptamersincorporate one or more deoxyinosine (deoxyriboinosine) nucleotides thatenable an endonuclease V enzyme to recognize the oligonucleotide aptameras a substrate and modify its structure by cleavage. Nucleotides in theaptamers may be modified at the phosphates, sugar moieties or nucleotidebases. The structural modifications can be “duplex-stabilizingmodifications.” “Duplex-stabilizing modifications” refer to structuralmodifications, the presence of which in double-stranded nucleic acidsprovides a duplex-stabilizing effect when compared in thermal stability,usually measured as “Tm,” with respective nucleic acid complexes thathave no such structural modification and, e.g., comprise naturalnucleotides. Duplex-stabilizing modifications are structuralmodifications that are most commonly applied in synthesis of probes andprimers, as represented by modified nucleotides and ‘tails’ likeintercalators and minor groove binders as, for example, disclosed inU.S. Pat. No. 8,349,556 to Kutyavin, I. V.; U.S. Pat. No. 7,794,945 toHedgpeth, J. et al.; U.S. Pat. No. 6,127,121 to Meyer, Jr., R. B., etal.; U.S. Pat. No. 5,801,155 to Kutyavin, I. V., et al., and thereferences cited in. Duplex-stabilizing modifications can be used toprepare aptamers of the invention, for example, to improve thermalstability of stem (duplex) structures of hairpin-like aptamers. Inpreferred methods of the invention, the oligonucleotide aptamers aremodified in (e.g., during) DNA synthesis reactions using enzymaticactivity of one or more aptamer-modifying enzyme(s). In this aspect, theterms “modify,” “modification,” and “structural modifications” meanchanges in the initial chemical structure of the aptamers. The change istriggered by Endonuclease V enzymes, resulting in a cleavage of one ormore phosphodiester bonds. In the methods of the invention, theseEndonuclease V-triggered structural modifications reduce or eliminatethe ability of the oligonucleotide aptamer to bind to the DNA polymeraseand block or reduce its activity in the reaction. In certain aspects,oligonucleotide aptamers comprise one or more deoxyinosine nucleotides,and aptamer modification results from aptamer cleavage (phosphodiesterbond cleavage) by endonuclease V, at the second phosphodiester bond inthe DNA strand on the 3′ side of a deoxyinosine nucleotide.

In particular aspects, the term “secondary structure” refers to anintramolecular complex formation of one sequence in a poly- oroligonucleotide with another sequence in the same polymer due tocomplete or partial complementarity between these two sequences formedbased on the principal rules of Watson-Crick base pairing. The terms“hairpin” structure and “stem-loop” structure as referred to hereindescribe elements of secondary structure, and both terms refer to adouble-helical region (stem) formed by base pairing betweencomplementary sequences within a single strand RNA or DNA.

As used herein, the term “nuclease” refers to an enzyme that expresses aphosphomonoesterase or phosphodiesterase activity and is capable ofcleaving a phosphoester bond in compounds such as R′—O—P(O)(OH)₂ andR′—O—P(O)(OH)—O—R,″ resulting in products R′—OH+P(O)(OH)₃ andR′—OH+P(O)(OH)₂—O—R″ (or R″—OH+P(O)(OH)₂—O—R′), respectively, andwherein R′ and R″ may be moieties of any structure which are notnecessarily of a nucleotide nature. The term “nucleases” incorporatesboth “exo” and “endo” nucleases. In certain aspects, endonuclease V isused to cleave phosphodiester bonds of oligonucleotide aptamerscomprising one or more deoxyinosine nucleotides.

The term “aptamer-modifying enzymatic activity” refers to anaptamer-modifying enzyme or mixture of aptamer-modifying enzymes whichrecognize an aptamer of the invention as a substrate and modifies itsstructure so that the inhibitory activity of the aptamer issubstantially disabled. In particular aspects, this recognition isdirected by presence of one or more deoxyinosine nucleotides within theaptamer structure. These aptamer structural modifications caused byendonuclease V enzymatic activity reduce the ability of theoligonucleotide aptamer to bind to the DNA polymerase and to block orreduce the polymerase activity in the reaction mixture.

As used herein, the term “deoxyinosine” refers to deoxyinosine andstructural modifications of deoxyinosine, including but not limited to7-deaza-deoxyinosine, 8-aza-7-deaza-deoxyinosine and other structuralmodifications of deoxyinosine, and including various ribonucleotidederivatives thereof, which can be recognized and cleaved by anendonuclease V enzymatic activity.

As used herein, the term “deoxyuridine” refers to deoxyuridine andstructural modifications of deoxyuridine, including variousribonucleotide derivatives thereof, that can be recognized and cleavedby an endonuclease V enzymatic activity.

As used herein, the term “endonuclease V” refers to repair enzymes thatrecognize deoxyinosine in DNA, and hydrolyze the second phosphodiesterbond in the DNA strand on the 3′ side of a deoxyinosine nucleotide (see,e.g., U.S. Pat. No. 8,143,006 to Kutyavin, I. V.).

The term “DNA polymerase” refers to an enzyme that catalyzes synthesisof deoxyribonucleic acids (DNAs), most commonly double-stranded DNAs,using single-stranded DNAs as “templates.” The DNA synthesis is usuallyinitiated by an oligonucleotide primer that is hybridized to acomplementary template strand. Starting from this template-hybridizedprimer, DNA polymerase creates a Watson-Crick complementary strand inthe presence of 2′-deoxyribonucleotide 5′-triphosphates (dNTPs). Theterm “DNA polymerase,” as used herein, also incorporates “reversetranscriptases,” enzymes which can perform DNA synthesis usingsingle-stranded ribonucleic acids (RNAs) as template strands.

“Polynucleotide” and “oligonucleotide” are used herein interchangeablyand in each case means a linear polymer of nucleotide monomers.Polynucleotides typically range in size from a few monomeric units,e.g., 5-60, when they are usually referred to as “oligonucleotides,” toseveral thousand monomeric units. The exact size will depend on manyfactors, which in turn depends on the ultimate function or use of theoligonucleotide. The oligonucleotides may be generated in any manner,including chemical synthesis, DNA replication, reverse transcription, ora combination thereof. Unless otherwise specified, whenever apolynucleotide or oligonucleotide is represented by a sequence ofletters, for example, “TTCTTAGCGTTT (SEQ ID NO:21),” it is understoodherein, unless otherwise specified in the text, that the nucleotides arein 5′→3′ order from left to right and that “A” denotes deoxyadenosine,“C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotesdeoxythymidine. Usually DNA polynucleotides comprise these fourdeoxyribonucleosides linked by phosphodiester linkage whereas RNAcomprises uridine (“U”) in place of “T” for the ribose counterparts.

The terms “oligonucleotide primer” and/or “primer” refer to asingle-stranded DNA or RNA molecule that hybridizes to a complementarytarget nucleic acid and serves to prime enzymatic synthesis of a secondnucleic acid strand in the presence of a DNA polymerase. In this case,the target nucleic acid “serves as a template” for the oligonucleotideprimer.

In particular aspects, the terms “complementary” or “complementarity”are used herein in reference to the polynucleotide base-pairing rules.Double-stranded DNA, for example, consists of base pairs wherein, forexample, G complexes or pairs with C via formation of a three hydrogenbond complex, and A complexes or pairs with T via formation of a twohydrogen bond complex, such that G is regarded as being complementary toC and A is regarded as being complementary to T. In this sense, forexample, an oligonucleotide 5′-GATTTC-3′ is complementary to thesequence 3′-CTAAAG-5′ via intrastrand G:C and A:T hydrogen bondinginteractions. Complementarity may be “partial” or “complete.” In partialcomplementarity, only some of the nucleic acids bases are matchedaccording to the base pairing rules. “Complementarity” may also be usedin reference to individual nucleotides and oligonucleotide sequenceswithin the context of polynucleotides (e.g., inter-strandcomplementarity). The terms “complementary” and “complementarity” referto the most common type of complementarity in nucleic acids, namely,Watson-Crick base pairing as described above, although theoligonucleotides may alternately participate in other types of“non-canonical” pairings like Hoogsteen, wobble and G-T mismatchpairing.

The term “natural nucleosides” refers to the four standard2′-deoxyribonucleosides (usually named herein as “deoxynucleosides” or“deoxyribonucleosides”) that are found in DNAs isolated from naturalsources. Natural nucleosides are deoxyadenosine, deoxycytidine,deoxyguanosine, and deoxythymidine. The term also encompasses theirribose counterparts, with uridine (U) in place of thymidine. The samename variations are applied herein to “natural nucleotides.”

As used herein, the terms “unnatural nucleosides” or “modifiednucleosides” refer to nucleoside analogs that are different in theirstructure from those natural nucleosides for DNA and RNA polymers. Somenaturally occurring nucleic acids contain nucleosides that arestructurally different from the natural nucleosides defined above, forexample, DNAs of eukaryotes may incorporate 5-methyl-cytosine, and tRNAscontain many nucleoside analogs. However, as used herein, the terms“unnatural nucleosides” or “modified nucleosides” encompasses thesenucleoside modifications even though they can be found in naturalsources.

The term “reaction mixture” generally means herein a solution containingall necessary reactants for performing DNA synthesis such as a DNApolymerase, oligonucleotide primer(s), template polynucleotide,deoxyribonucleoside 5′-triphosphates, reaction cofactors (e.g.,magnesium or manganese ions), etc. The reaction mixture can incorporateother reaction components that help to improve DNA synthesis (e.g.,buffering and salt components, detergents, proteins like bovine serumalbumin (BSA), scavengers, etc.) or components that are necessary fordetection of the newly synthesized DNA molecules such as, for example,fluorescent dyes and oligonucleotide probes. A reaction mixture isusually prepared at low temperatures at which enzymatic components areinactive, for example, by mixing the components on ice at ˜0° C. Whenthe reactions are ready, the mixtures can be heated to the desiredreaction temperatures. In this aspect, the term “reaction temperature”refers to a temperature or a temperature range at which DNA synthesis isperformed. In case of PCR reactions, it is usually taken as the lowestthermo-cycling temperature, commonly called the annealing temperature.

“dNTPs” is an abbreviation of a mixture of two or more of the fournatural deoxynucleoside 5′-triphosphates that are useful to facilitateprimer extension with a DNA polymerase and/or amplification.Respectively, the abbreviations “dATP,” “dCTP,” “dGTP,” and “dTTP”correspond to the individual nucleotides. In some embodiments, the fourdNTPs are present at equal concentrations. In other embodiments, theconcentrations of the dNTPs are not all identical. In some embodiments,fewer than all four dNTPs are present. For example, only one dNTP may bepresent, or a pair-wise combination, or three of four dNTPs may bepresent in the mixture.

In some aspects, “amplification” and “amplifying” deoxyribonucleicacids, in general, refer to a procedure wherein multiple copies of DNAof interest are generated. The DNA amplification can be performed at aconstant temperature using “isothermal amplification reactions.”Examples of isothermal amplification reactions include, but are notlimited to, Strand Displacement Amplification (SDA) (U.S. Pat. No.5,270,184 to Walker, G. T., et al.; U.S. Pat. No. 6,214,587 toDattagupta, N., et al.), Rolling Circle amplification (RCA) (U.S. Pat.No. 5,854,033 to Lizardi, P.), Loop-Mediated Amplification (LMA) (U.S.Pat. No. 6,410,278 to Notomi, T. and Hase, T.), isothermal amplificationusing chimeric or composite RNA/DNA primers (U.S. Pat. No. 5,824,517 toCleuziat, P. and Mandrand, B.; U.S. Pat. No. 6,251,639 to Kurn, N.),Nucleic Acid Sequence-Based Amplification (NASBA) (U.S. Pat. No.6,063,603 to Davey, C. and Malek, L. T.), and many other methods.

“PCR” is an abbreviation of “polymerase chain reaction,” anart-recognized nucleic acid amplification technology (e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202 to Mullis, K. B.). Commonly used PCRprotocol employs two oligonucleotide primers, one for each strand,designed such that extension of one primer provides a template for theother primer in the next PCR cycle. Generally, a PCR reaction consistsof repetitions (cycles) of (i) a denaturation step that separates thestrands of a double-stranded nucleic acid, followed by (ii) an annealingstep, which allows primers to anneal to positions flanking a sequence ofinterest on a separated strand, and then (iii) an extension step thatextends the primers in a 5′ to 3′ direction, thereby forming a nucleicacid fragment complementary to the target sequence. Each of the abovesteps may be conducted at a different temperature using an automatedthermocycler. The PCR cycles can be repeated as often as desiredresulting in an exponential accumulation of a target DNA fragment whosetermini are usually defined by the 5′-ends of the primers used. Althoughconditions of PCR can vary in a broad range, a double-stranded targetnucleic acid is usually denatured at a temperature of >90° C., primersare annealed at a temperature in the range of about 50-75° C., and theextension is preferably performed in a 72-75° C. temperature range. InPCR methods, the annealing and extension can be combined into one stage(i.e., using a single temperature). The term “PCR” encompasses itsnumerous derivatives such as “RT-PCR,” “real-time PCR,” “nested PCR,”“quantitative PCR,” “multiplexed PCR,” “asymmetric PCR,” and the like.

“Real-time detection” means an amplification reaction for which theamount of reaction product, i.e., target nucleic acid, is monitored asthe reaction proceeds. Real-time detection is possible when alldetection components are available during the amplification and thereaction composition and conditions support both stages of the reaction,the amplification and the detection.

As used herein, the term “kit” refers to any system for deliveringmaterials. In the context of reaction assays, such delivery systemsinclude elements allowing the storage, transport, or delivery ofreaction components such as oligonucleotides, buffering components,additives, reaction enhancers, and the like in the appropriatecontainers from one location to another commonly provided with writteninstructions for performing the assay. Kits may include one or moreenclosures or boxes containing the relevant reaction reagents andsupporting materials. The kit may comprise two or more separatecontainers wherein each of those containers includes a portion of thetotal kit components. The containers may be delivered to the intendedrecipient together or separately.

In general, the term “design” in the context of the methods has broadmeaning and in certain respects is equivalent to the term “selection.”For example, the terms “primer design” and “aptamer design” can mean orencompass selection of a particular oligonucleotide structure includingthe nucleotide primary sequence and structural modifications (e.g.,labels, modified nucleotides, linkers, etc.). In particular aspects, theterms “system design” and “assay design” relate to the selection of any,sometimes not necessarily to a particular, methods including allreaction conditions (e.g., temperature, salt, pH, enzymes, including theaptamer-modifying enzymes and DNA polymerase, oligonucleotide componentconcentrations, etc.), structural parameters (e.g., length and positionof primers and probes, design of specialty sequences, etc.), and assayderivative forms (e.g., post-amplification, real time, immobilized, FRETdetection schemes, etc.) chosen to amplify and/or to detect the nucleicacids of interest.

Reversible Blocking DNA Synthesis Activity of DNA Polymerases UsingAptamers.

Prior use of oligonucleotide aptamers during DNA synthesis haveattempted to block DNA polymerase activity, preferably completely, atlow temperatures, while releasing (activating) the DNA polymeraseactivity, preferably completely, at an elevated reaction temperature. Itis difficult, however, to achieve complete ‘block-and-release’ formatsusing conventional aptamer-based methods (e.g., Yakimovich, O. Yu., etal., (2003); Jayasena, S. D. (1999); U.S. Pat. No. 5,693,502 to Gold, L.and Jayasena, S. D. (1997)). Effective blockage of DNA polymerase at lowtemperatures commonly leads to ineffective release of the enzyme at theelevated reaction temperature and vice versa. Aspects of the presentinvention provide a solution to this long-standing problem in the art.As in the conventional approaches cited above, the DNA polymeraseactivity is blocked or reduced in methods of the invention by thepresence of an oligonucleotide aptamer that binds to the DNA polymerase,blocking the DNA synthesis activity of the DNA polymerase. Unlike priorart techniques, however, in methods of the invention, theaptamer-inactivated DNA polymerase is activated by providing to a DNAsynthesis reaction mixture an endonuclease V enzyme activity thatrecognizes the oligonucleotide aptamer as a substrate and cleaves itsstructure. This cleavage reduces or eliminates the binding of theoligonucleotide aptamer to the DNA polymerase and thereby reactivatesthe DNA synthesis activity of the DNA polymerase.

In some embodiments of the invention, activation of anaptamer-inactivated DNA polymerase in a reaction mixture, comprising (i)a DNA polymerase, (ii) oligonucleotide aptamer in an amount effective toinhibit the DNA synthesis activity of the DNA polymerase, (iii) anendonuclease V aptamer-modifying enzyme and other components necessaryfor DNA synthesis, is facilitated using a reaction temperature thataccelerates (or facilitates) both DNA polymerase and endonuclease Venzymatic activities. For example, the reaction mixture can be preparedat low temperature (first temperature) at which a DNA polymerase iseffectively blocked by an aptamer and an aptamer-modifying enzyme,endonuclease V, has sufficiently reduced or preferably no activity(e.g., at 0° C.), and then the activation of the aptamer-inactivated DNApolymerase is facilitated by heating the reaction to a temperature(second temperature) that accelerates or facilitates theaptamer-modifying endonuclease V enzymatic activity. If necessary, a DNApolymerase can be activated by the endonuclease V enzyme(s) at anytemperature below the reaction temperature for DNA synthesis. This canbe applied, for example, when a particular endonuclease V enzyme isunstable at the reaction temperature for DNA synthesis, for example, dueto denaturation. In this case, the DNA polymerase is first activated atan intermediate temperature wherein the endonuclease V is active andthen heated to the reaction temperature to perform DNA synthesis.

In some aspects, the reaction mixture is created by addition of aqueoussolution to one or more reaction components which are initially in adried state as disclosed, for example, in U.S. Pat. No. 3,721,725 toBriggs, A. R. and Maxwell, T. J. (1973) (incorporated herein byreference). For example, in some methods of the invention for DNAamplification and detection, the aqueous solution can be a samplesolution or solution that contains one or more polynucleotide templatesfor DNA synthesis whereas all other reaction components such as the DNApolymerase, aptamer, endonuclease V enzymatic activity, dNTPs, catalyticcofactors such as magnesium (Mg2+) and/or manganese (Mn2+) ion (e.g.,provided as a chloride salt), buffering components, detergents, proteinslike bovine serum albumin (BSA), scavengers, etc., are initiallyprovided in a dry state.

In some aspects, DNA synthesis results in DNA amplification in thereaction mixture. The DNA amplification can be an isothermalamplification reaction, for example, as described in U.S. Pat. No.5,270,184 to Walker, G. T., et al.; U.S. Pat. No. 6,214,587 toDattagupta, N., et al.; U.S. Pat. No. 5,854,033 to Lizardi, P.; U.S.Pat. No. 6,410,278 to Notomi, T. and Hase, T.; U.S. Pat. No. 5,824,517to Cleuziat, P. and Mandrand, B.; U.S. Pat. No. 6,251,639 to Kurn, N.;U.S. Pat. No. 6,063,603 to Davey, C. and Malek, L. T., and many othermethods. In other aspects, the DNA amplification can be a PCR reaction(e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis, K. B.). Inmethods of the invention, the DNA amplification is performed fordetection as well as measuring an amount of a target DNA in the reactionmixture.

As has been established in the art (e.g., Yao, M., Kow, Y. W., 1997), anumber of nucleotide modifications can be recognized by the endonucleaseV enzymes to provide for the oligonucleotide cleavage. For example, anoligonucleotide aptamer that contains one or more deoxyuridinenucleotide(s) as described, for example, in Yao, M., Kow, Y. W., 1997,can be used in methods, compositions and kits of the present invention,such that contact of such aptamers with an endonuclease V enzymaticactivity causes cleavage of the deoxyuridine-containing aptamer.However, the preferred modified nucleotide in aptamers is one or moredeoxyinosine nucleotide(s). The endonuclease V enzymes of the presentinvention can cleave single-stranded and double-strandedoligonucleotides. Therefore the aptamers of the present invention can beboth, single and double-stranded. In particular embodiments, theaptamers of the invention are stem-loop or hairpin-like molecules.Effective use of these two exemplary combinations (endonucleaseV)+(deoxyinosine-incorporating aptamers) is illustrated in the Examplesprovided herein using, in particular, hairpin-like structures (FIGS.2-7; as described in working Example 2 herein below). Deoxyinosinenucleotide can be located either in loop or stem fragments of thehairpin-like aptamers of the invention. Preferred locations ofdeoxyinosine modifications in aptamers of the present invention aredescribed herein.

The oligonucleotide aptamers as well as oligonucleotide primers andprobes can be prepared by any method of oligonucleotide synthesisselected by a person of ordinary skill in the art, such as methods thatutilize (2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidites (Example 1).Protected nucleotides and derivatives, linkers, dyes, tails, solidsupports, and other appropriate components can be prepared by methods oforganic chemistry or obtained from market providers such as, forexample, Glen Research and Biosearch Technologies. Suppliers such asIntegrated DNA Technologies and Biosearch Technologies also offeroligonucleotide custom synthesis including numerous structuralmodifications such as deoxyinosine and deoxyuridine. In the methods ofthe invention, selection of an aptamer structure, including one or moredeoxyinosine nucleotides, is intended to achieve (i) completedeactivation of the DNA polymerase at the initial reaction assemblytemperature, (ii) complete or substantially complete (or as much aspossible) deactivation of the DNA polymerase at the elevated reactiontemperature for DNA synthesis, and (iii) substantially complete orcomplete (or as much as possible) reactivation of this enzyme at the DNAsynthesis reaction temperature once the aptamer has been modified by theendonuclease V enzymatic activity. Generally, endonuclease V enzymes donot interfere with DNA synthesis (primer extension) or DNAamplification. The location and number of deoxyinosine nucleotideswithin an aptamer, as well as the rate and efficiency of theendonuclease V enzymes is preferably taken into consideration.Preference is given to deoxyinosine locations within an aptamer thathave little or no negative effect on stability of the aptamer-DNApolymerase complex, but sufficiently disturb the structural integrity ofthe aptamer so that the cleaved aptamer does not bind to or inhibit theDNA polymerase. Results from aptamers containing deoxyinosinenucleotides (SEQ ID NOS:6-10 and 12-20) as the endonucleaseV-recognition motif are illustrated in FIGS. 3, 5, 7, and 9.Surprisingly, even a single base modification at numerous locationsshowed excellent results in the exemplary assays.

According to the prior art (Yakimovich, O. Yu., et al. (2003); Jayasena,S. D. (1999); U.S. Pat. No. 5,693,502 to Gold, L. and Jayasena, S. D.),efficiency of hairpin-type aptamer binding to DNA polymerase isdetermined by (i) a loop segment, which is a substantially conservativesequence, and (ii) length of the stem duplex, which preferably needs tobe ˜19-20 base pairs or longer. In addition, the present inventors havefound that the sequence of the stem fragment can be another importantfactor affecting the stability of an aptamer-polymerase complex. Forexample, the stem sequence of aptamers SEQ ID NOS:5 and 11 used in thepresent working Examples mostly comprises a (AGT)₅ nucleotide repeat(see FIGS. 2 and 4). However, these aptamers as well as many derivatives(SEQ ID NOS:6-10 and 12-20) were very effective in blocking not only Taq(FIGS. 3, 5, and 7), but also many other DNA polymerases (FIG. 9). Outof six DNA polymerases investigated, only Bst polymerase (largefragment) was not inactivated by the aptamer SEQ ID NO:6. Analysis ofFIG. 9 points to additional surprising results. First, regardless of thedifference in reaction temperature, aptamer SEQ ID NO:6 blocked Phusion®polymerase much more efficiently at 65° C. than Taq polymerase at 60° C.According to additional surprising aspects of the invention, therefore,the best-blocking sequence of the aptamer hairpin duplex may be somewhatpolymerase-specific. Second, the duplex sequence in an aptamer of theinvention can be further optimized by base pair changes for even betterpolymerase blockage in each particular case. Third, using the sequenceof aptamers SEQ ID NOS:5 and 11 as an origin, sequence optimization forstrongest binding can be performed for every DNA polymerase known in theart, although for some DNA polymerases like Bst, optimization mayrequire alterations in the loop segment of the aptamer as well as in thestem. In this sense, the present disclosure also provides methods ofscreening for more optimal aptamers for use in the disclosed methods.

Aptamers of the invention, whether single-stranded or double-stranded,can contain any number of modified nucleotides, internal and externallinker and moieties and other structural modifications as long as thesemodifications do not interfere with the DNA polymerase deactivation andthen the activation processes during DNA synthesis. For example, ifdesirable in a specific assay, aptamers of the invention can includephosphorothioate bonds at their termini to protect the aptamers from theexonuclease hydrolysis (Skerra, A., 1992). Hairpin-type aptamers of theinvention can also contain non-complementary 5′ or 3′ nucleotidesequences. Preference should be given to structural modifications thathelp to deactivate the DNA polymerase and do not adversely affect theendonuclease V enzyme activation reaction. Both loop and stem fragmentscan be modified in the hairpin-type aptamers. Although the loop segmentsdescribed in Yakimovich, O. Yu., et al. (2003), Jayasena, S. D. (1999),and U.S. Pat. No. 5,693,502 to Gold, L. and Jayasena, S. D. containconserved sequence motifs, the results in FIG. 7 (Example 2) herein showthat deoxyinosine substitution(s) at certain loop positions have verylittle (SEQ ID NO:20) or no effect (SEQ ID NOS:17 and 19) on aptamerperformance (FIG. 7).

Methods of the invention can be performed using one or more reactiontemperatures wherein a DNA polymerase and endonuclease V expresssuitable activity. Specificity of DNA synthesis is usually increased athigher temperatures, and therefore thermostable enzymes are usuallypreferred. The upper level of the reaction temperature can be selectedbased on the DNA polymerase stability. In cases when an endonuclease Vis not stable at a desired reaction temperature, the DNA polymeraseactivation can be initiated at a lower intermediate temperature whereinthe endonuclease V is stable and active and then raised to the desiredreaction temperature for DNA synthesis (primer extension). In someembodiments, a DNA polymerase is preferably first deactivated bycontacting (e.g., by combining or mixing) with an aptamer before otherreaction components of the DNA synthesis are added. Molar reactionconcentration of an aptamer applied should be at least equal to theconcentration of a DNA polymerase or preferably greater. Marketproviders commonly do not disclose the molar amount of the enzymes,therefore the precise excess of the aptamers over the DNA polymerasesused in Examples provided herein was not known. However, the aptamersused in the Examples below were estimated to be present in a range of˜10-40 fold, or greater, molar excess relative to DNA polymerase. Insome embodiments, the aptamer is present in a molar excess (ratio) overthe DNA polymerases of at least ˜5-fold, although the ratio can behigher or lower than 5-fold. The amounts of the enzymes, aptamers andother reaction components used in the reaction may be optimized anddepend on many factors including, but not limited to selection of theparticular enzymes, enzymatic activities at the reaction temperature,reaction temperature itself, nature of the aptamers, and their specialendonuclease V recognition motifs (e.g., deoxyinosine or deoxyuridine,etc.) to provide for cleavage of the aptamers. Methods of the presentinvention can be particularly useful for so-called ‘fast’ PCR with acycle time shorter than 20 seconds.

In certain embodiments, methods of the invention can be practiced usinga kit comprising a DNA polymerase-binding oligonucleotide aptamerrecognizable and modifiable by an endonuclease V enzymatic activity, andan endonuclease V enzyme activity to provide for specific cleavage ofthe aptamer. The kit can also include a DNA polymerase that is initiallydeactivated by the oligonucleotide aptamer. Alternatively, the kit caninclude, in addition to the endonuclease V enzyme activity, a complex ofthe DNA polymerase with the oligonucleotide aptamer, wherein thecomponents of this complex are present at a specific and optimal molarratio. As a matter of convenience, such a kit can include componentsallowing the storage, transport and other reaction components such asoligonucleotides, buffering components, additives, reaction enhancers,etc. The aptamers of the kits can be single-stranded or have a stem-loopstructure, and they can incorporate one or more deoxyinosines ordeoxyuridines. The kits can be used for the DNA synthesis, amplificationas well as the detection of the amplified DNA fragments.

In some embodiments, the invention includes a reaction mixturecomprising a DNA polymerase, an oligonucleotide aptamer that binds tothe DNA polymerase and present in an amount effective to inhibit DNAsynthesis activity of the DNA polymerase, an endonuclease V enzymeactivity that is capable of cleaving the oligonucleotide aptamer toreduce or eliminate binding of the oligonucleotide aptamer to the DNApolymerase, and other reaction components necessary for DNA synthesis isalso a subject of the present invention. In some embodiments, thereaction mixture can be assembled using concentrated stock solutions ofone or more components, usually in water to provide the desiredcomponent concentration in the final mixture. Mixing is recommended tobe performed at a low temperature (e.g., close to 0° C.) at which theenzymes, particularly endonuclease V enzymes, are inactive. Preferably,the reaction mixture should be used for DNA synthesis soon afterpreparation. Storage of a fully assembled reaction mixture is notrecommended. However, reaction components including enzymes can retainactivity for a long time (days, months, or even years) in a dried state.For example, in some embodiments of the invention, one or more of thecomponents for forming a mixture of the invention can be provided in adried form, such as dried beads as described, for example, in U.S. Pat.No. 3,721,725 to Briggs, A. R. and Maxwell, T. J. (1973), including (butnot limited to) DNA polymerase, oligonucleotide aptamer, andendonuclease V enzyme activity such that one or more of the componentsis prepared in a form of dried beads as described, for example, in U.S.Pat. No. 3,721,725 to Briggs, A. R. and Maxwell, T. J. (1973). In someembodiments, the mixture comprises DNA polymerase, an oligonucleotideaptamer that binds to the DNA polymerase and present in an amounteffective to inhibit DNA synthesis activity of the DNA polymerase, andan endonuclease V enzyme activity that is capable of cleaving theoligonucleotide aptamer to reduce or eliminate binding of theoligonucleotide aptamer to the DNA polymerase, which are mixed togetherin a dried form.

Example 1 Synthesis of Aptamers, Primers and Fluorescent Probes

Standard phosphoramidites, including modified nucleotide analogs such asdeoxyinosine phosphoramidite (Catalog Number: 10-1040-xx), solidsupports and reagents to perform solid support oligonucleotidesynthesis, were purchased from Glen Research. A 0.25 M5-ethylthio-1H-tetrazole solution was used as a coupling agent.Oligonucleotides were synthesized either on ABI394 DNA synthesizer(Applied Biosystems) or MerMaid 6 DNA synthesizer (BioAutomationCorporation) using protocols recommended by the manufacturers for 0.2μmole synthesis scales. Fluorescein (FAM) conjugated to 5-position ofdeoxyribouridine (U) of probe SEQ ID NO:29 (FIG. 8) was introduced tothe hairpin during oligonucleotide synthesis using5′-dimethoxytrityloxy-5-[N-((3′,6′-dipivaloylfluoresceinyl)-aminohexyl)-3-acrylimido]-2′-deoxyribouridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research, Catalog Number: 10-1056-xx). A 6-fluorescein reportingdye was incorporated onto the 5′-end, and a BHQ1 quencher was introducedto the 3′-end of probe SEQ ID NO:3 (FIG. 1) using respectivephosphoramidite and CPG from Biosearch Technologies (Catalog numbers:BNS-5025 and BG1-5041G). After the automated synthesis, oligonucleotideswere deprotected in aqueous 30% ammonia solution by incubation for 12hours at 55° C. or 2 hours at 70° C.

Tri-ON oligonucleotides were purified by HPLC on a reverse phase C18column (LUNA 5 μm, 100 A, 250×4.6 mm, Phenomenex Inc.) using gradient ofacetonitrile in 0.1 M triethyl ammonium acetate (pH 8.0) or carbonate(pH 8.5) buffer with flow rate of 1 ml/min. A gradient profile includingwashing stage 0→14% (10 sec), 14→45% (23 min), 45→90% (10 min), 90→90%(5 min), 90→0% (30 sec), 0→0% (7 min) was applied for purification ofall Tri-ON oligonucleotides. The product containing fractions were drieddown in vacuum (SPD 1010 SpeedVac, TermoSavant) and trityl groups wereremoved by treatment in 80% aqueous acetic acid at room temperature for40-60 min. After addition to the detritylation reaction (100 μl) of 20μl sodium acetate (3 M), the oligonucleotide components wereprecipitated in alcohol (1.5 ml), centrifuged, washed with alcohol anddried down. Concentration of the oligonucleotide components wasdetermined based on the optical density at 260 nm and the extinctioncoefficients calculated for individual oligonucleotides using on-lineOligoAnalyzer 3.0 software provided by Integrated DNA Technologies.Based on the measurements, convenient stock solutions in water wereprepared and stored at −20° C. for further use. The purity of allprepared oligonucleotide components was confirmed by analytical 8-20%PAAG electrophoresis, reverse phase HPLC and by spectroscopy on Cary4000 UV-VIS spectrophotometer equipped with Cary WinUV software, BioPackage 3.0 (Varian, Inc.).

Example 2 Application of Deoxyinosine-Containing Aptamers to ControlPolymerase Activity of Taq Polymerase

This working example shows application of deoxyinosine-containingaptamers to control activity of Taq polymerase during PCR.

For the results shown in FIGS. 3, 5, and 7, reaction mixtures (25 μL)were prepared on ice by mixing corresponding stock solutions to provide200 nM forward primer (FIG. 1, SEQ ID NO:1), 300 nM reverse primer (SEQID NO:2), 200 nM FRET probe (SEQ ID NO:3), 0.02 U/μL Taq DNA polymerase(GenScript cat no: E00007), dNTPs (200 μM each), bovine serum albumin(0.1 μg/μL), 100 ng of human genomic DNA (GenScript cat no: M00094) and,when present, one of the aptamers SEQ ID NOS:5-20 (20 nM) in 5 mM MgCl₂,50 mM KCl, 20 mM Tris-HCl (pH8.0). The reaction tubes were quicklytransferred into SmartCycler instrument (Cepheid Corporation) andtemperature cycling initiated. The PCR time/temperature profilecomprised initial incubation at 95° C. for 15 seconds followed by 50cycles of incubation at 95° C. for 1 second and then at 60° C. for 20seconds. The reaction fluorescence was measured in every PCR cycleduring the annealing/extension stage (60° C.) and the results are shownin FIGS. 3, 5, and 7. Each fluorescence curve is an average of fouridentical reactions. Initial background fluorescence was subtracted bythe instrument software.

The reaction conditions used to generate the fluorescence profiles shownin FIGS. 3, 5, and 7 were identical except for the presence or absenceof endonuclease V enzymatic activity and the presence or absence ofdifferent oligonucleotides as potential inhibitors of polymeraseactivity. PCR reactions were performed either in the absence (leftdiagram of each figure) or presence (right diagram) of T. maritimaendonuclease V (0.04 U/μL, Fisher Scientific cat no: FEREN0141) in thereaction mixtures. Structures of the oligonucleotide aptamers used inthe experiments of FIGS. 3, 5, and 7 are shown in FIGS. 2, 4, and 6,respectively.

In summary of this working Example, the real-time curves shown in FIGS.3 and 5 show that unmodified aptamer SEQ ID NO:5 and it structuralanalog SEQ ID NO:11 effectively block Taq polymerase activity during PCRand are not affected by the presence of endonuclease V activity in thereaction mixture. Analysis of the real-time PCR curves in FIGS. 3 and 5leads to the following conclusions. Virtually ideal results of DNApolymerase inactivation-activation were obtained for the aptamers SEQ IDNOS:6, 9, 12, and 13, wherein endonuclease V cleavage takes place nearbythe duplex center, but closer to the loop fragment. However, when thedeoxyinosine nucleotide is too close to the loop, the correspondingaptamer SEQ ID NO:7 becomes less effective in blockage of the DNApolymerase, likely due to profound destabilization of the aptamer duplexsegment adjacent to the loop. When the endonuclease V cleavage site islocated closer to the aptamer duplex 3′ or 5′ ends, as in the cases ofaptamers SEQ ID NOS:10 and 14, the truncated hairpin products werelikely to retain thermal stability and duplex length that is sufficientfor blockage of the Taq DNA polymerase during PCR. Moreover, thetruncated hairpin product resulted after the cleavage of aptamer SEQ IDNO:10 can be extended by the DNA polymerase thereby restoring theoriginal aptamer duplex structure. This may explain the extreme “noactivation” result observed for SEQ ID NO:10 (FIG. 3B). Incorporation ofmore than one deoxyinosine modifications into aptamer duplex (aptamersSEQ ID NOS:8, 15, and 16) can be applied, but it is generallyunnecessary. Furthermore, increase in the number of I-C base pairsusually leads to destabilization of the aptamer duplex and thusnegatively affects ability of the aptamers to inactivate the DNApolymerase (e.g., aptamers SEQ ID NOS:8 and 16).

Regarding ability of the hairpin-like aptamers to inactivate Taq DNApolymerase, the loop sequence 5′TTCTTAGCGTTT3′ (SEQ ID NO:21) is knownto be highly conserved (e.g. Yakimovich, O. Yu., et al. (2003),Jayasena, S. D. (1999), and U.S. Pat. No. 5,693,502 to Gold, L. andJayasena, S. D.). Nevertheless, a number of the nucleotide substitutionsby deoxyinosine in the aptamers SEQ ID NOS:17-20 shown in FIG. 6 wasinvestigated, and the real-time PCR results are provided in FIG. 7.Significant difference in properties was found for the aptamers SEQ IDNOS:17 and 18. Both aptamers completely lose capability to block Taq DNApolymerase after endonuclease V cleavage. However, in the absence ofsuch cleavage, the aptamer SEQ ID NO:18 showed very weak inactivation ofthe DNA polymerase whereas the aptamer SEQ ID NO:17 appeared to be idealin the polymerase inactivation-activation performance. The aptamers SEQID NOS:17 and 18 represent a homologous guanine-to-hypoxanthine purinesubstitution. The most surprising results were obtained for the aptamersSEQ ID NOS:19 and 20 representing nonhomologous (pyrimidine-purine)thymine-hypoxanthine base alteration within the highly conservative loopsequence. Both aptamers inactivated the DNA polymerase (FIG. 7A),although the aptamer SEQ ID NO:20 was somewhat less effective, whereastreatment of the PCR reactions with endonuclease V completely restoredTaq DNA polymerase activity. In conclusion, investigation of thedeoxyinosine substitutions within the aptamer loop sequences allowedidentification of at least three exemplary nucleotide locations that canbe used in embodiments of the present invention. For example, the loopsequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCTIAGCGTTT-3′ (SEQ IDNO:23), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:24), 5′-TTCIIAGCGTTT-3′ (SEQ IDNO:25), 5′-TTCITAICGTTT-3′ (SEQ ID NO:26), 5′-TTCTIAICGTTT-3′ (SEQ IDNO:27), and 5′-TTCIITAICGTTT-3′ (SEQ ID NO:28) incorporating 1, 2, or 3deoxyinosine nucleotides can be used in design of the hairpin-likeaptamers for the methods of the present invention.

Example 3 Kinetics of Activation by Endonuclease V of Various DNAPolymerases Initially Blocked by Deoxyinosine-Containing Aptamer

This working example shows the kinetics of activation by endonuclease Vof Taq (GenScript cat no: E00007), Q5® (New England Biolabs cat no:M0491S), Vent® (New England Biolabs cat no: M0254S), Deep Vent® (NewEngland Biolabs cat no: M0258S), Bst large fragment (New England Biolabscat no: M0275S), and Phusion® (New England Biolabs cat no: M0530S) DNApolymerases initially blocked by a deoxyinosine-containing aptamers.

For FIG. 9, reaction mixtures (25 μL) were prepared on ice by mixingcorresponding stock solutions to provide self-priming hairpin SEQ IDNO:29 (200 nM, FIG. 8), a DNA polymerase (0.008 U/μL), dNTPs (200 μMeach), bovine serum albumin (0.1 μg/μL) and, when present, the aptamerSEQ ID NO:6 (80 nM, FIG. 2) and T. maritima endonuclease V (0.02 U/μL,Fisher Scientific cat no: FEREN0141) in 5 mM MgCl₂, 50 mM KCl, 20 mMTris-HCl (pH8.0). During preparation of the reaction mixture, theself-priming hairpin (SEQ ID NO:29) and endonuclease V were always addedlast to a premixed solution. Then the reaction tubes were transferredinto a SmartCycler instrument (Cepheid Corporation) and heated to 60 or65° C. as indicated for each fluorescence profile in FIG. 9. Thereaction fluorescence was monitored every 7 seconds. The plotted curvesare the averages of four paralleled identical reactions. Initialbackground fluorescence was subtracted.

The results of FIG. 9 show that not only Taq polymerase, but also manyother DNA polymerases can be inactivated and then activated usingendonuclease V-cleavable aptamers of the present invention. Only one ofsix investigated exemplary DNA polymerases, particularly Bst DNApolymerase, was not inactivated by aptamer SEQ ID NO:6. Other DNApolymerases showed a ‘delayed’ activation in the presence of theendonuclease V before the DNA synthesis activity was restored.

References cited, and incorporated by reference herein for theirrespective teachings:

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1-16. (canceled)
 17. A kit for activating an aptamer-inactivated DNApolymerase, comprising: an endonuclease V enzymatic activity; and a DNApolymerase-binding oligonucleotide aptamer cleavable by an endonucleaseV enzymatic activity.
 18. The kit of claim 17, wherein theoligonucleotide aptamer comprises one or more deoxyinosine nucleotides.19. The kit of claim 17, wherein the oligonucleotide aptamer comprises astem-loop structure.
 20. The kit of claim 19, wherein the one or moredeoxyinosine nucleotides are located in the stem segment of thestem-loop structure.
 21. The kit of claim 19, wherein the one or moredeoxyinosine nucleotides are located in the loop segment of thestem-loop structure.
 22. The kit of claim 21, wherein the loop of thestem-loop structure, comprises a nucleotide sequence selected from thegroup consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:22),5′-TTCTIAGCGTTT-3′ (SEQ ID NO:23), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:24),5′-TTCIIAGCGTTT-3′ (SEQ ID NO:25), 5′-TTCITAICGTTT-3′ (SEQ ID NO:26),5′-TTCTIAICGTTT-3′ (SEQ ID NO:27), and 5′-TTCIITAICGTTT-3′ (SEQ IDNO:28).
 23. The kit of claim 22, wherein the loop of the stem-loopstructure comprises one of the nucleotide sequences 5′-TTCITAGCGTTT-3′(SEQ ID NO:22), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:23), or 5′-TTCTTAICGTTT-3′(SEQ ID NO:24).
 24. The kit of claim 17, wherein the endonuclease Venzymatic activity comprises Thermotoga maritima endonuclease Venzymatic activity.
 25. A reaction mixture for use in a method of DNAsynthesis, which reaction mixture comprises: a DNA polymerase; anendonuclease V-cleavable oligonucleotide aptamer that binds reversiblyto the DNA polymerase, wherein the oligonucleotide aptamer is present inan amount effective to inhibit DNA synthesis activity of the DNApolymerase in the reaction mixture; and an endonuclease V enzymaticactivity capable of cleaving the oligonucleotide aptamer to reduce oreliminate binding of the oligonucleotide aptamer to the DNA polymerase,thereby activating or increasing the DNA synthesis activity of the DNApolymerase.
 26. The reaction mixture of claim 25, wherein the DNApolymerase activity and/or the endonuclease V enzymatic activity istemperature-dependent.
 27. The reaction mixture of claim 26, wherein theendonuclease V enzymatic activity increases when the reaction mixture isheated from a first temperature value to a second temperature value thatpromotes the endonuclease V enzymatic activity.
 28. The reaction mixtureof claim 25, wherein the DNA polymerase, oligonucleotide aptamer, andendonuclease V enzymatic activity are in a dried state.
 29. The reactionmixture of claim 25, wherein the oligonucleotide aptamer comprises oneor more deoxyinosine nucleotides.
 30. The reaction mixture of claim 29,wherein the oligonucleotide aptamer comprises a stem-loop structure. 31.The reaction mixture of claim 30, wherein the one or more deoxyinosinenucleotides are located in the stem segment of the stem-loop structure.32. The reaction mixture of claim 30, wherein the one or moredeoxyinosine nucleotides are located in the loop segment of thestem-loop structure.
 33. The reaction mixture of claim 32, wherein theloop of the stem-loop structure, comprises a nucleotide sequenceselected from the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:22),5′-TTCTIAGCGTTT-3′ (SEQ ID NO:23), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:24),5′-TTCIIAGCGTTT-3′ (SEQ ID NO:25), 5′-TTCITAICGTTT-3′ (SEQ ID NO:26),5′-TTCTIAICGTTT-3′ (SEQ ID NO:27), and 5′-TTCIITAICGTTT-3′ (SEQ IDNO:28).
 34. The reaction mixture of claim 33, wherein the loop of thestem-loop structure comprises one of the nucleotide sequences5′-TTCITAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:23), or5′-TTCTTAICGTTT-3′ (SEQ ID NO:24).
 35. The reaction mixture of claim 25,wherein the endonuclease V enzymatic activity comprises Thermotogamaritima endonuclease V enzymatic activity.
 36. The reaction mixture ofclaim 25, wherein the mixture further comprises one or more of dATP,dCTP, dGTP, and/or dTTP, and/or Mg²⁺ ion.
 37. An oligonucleotideaptamer, comprising a nucleic acid sequence that forms a hairpinstructure, having a stem and a loop portion, that binds to a DNApolymerase, wherein the stem and/or the loop portion comprises one ormore deoxyinosine nucleotides, and wherein the loop portion comprises anucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:21) that may besubstituted with deoxyinosine at one or more of positions 4, 5, and 7.38. The oligonucleotide aptamer of claim 37, wherein the loop portioncomprises the nucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:21). 39.The oligonucleotide aptamer of claim 37, wherein the one or moredeoxyinosine nucleotides are located in the stem segment of thestem-loop structure.
 40. The oligonucleotide aptamer of claim 37,wherein the one or more deoxyinosine nucleotides are located in the loopsegment of the stem-loop structure.
 41. The oligonucleotide aptamer ofclaim 40, wherein the loop of the stem-loop structure, comprises anucleotide sequence selected from the group consisting of5′-TTCITAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:23),5′-TTCTTAICGTTT-3′ (SEQ ID NO:24), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:25),5′-TTCITAICGTTT-3′ (SEQ ID NO:26), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:27),and 5′-TTCIITAICGTTT-3′ (SEQ ID NO:28).
 42. The oligonucleotide aptamerof claim 41, wherein the loop of the stem-loop structure comprises oneof the nucleotide sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:22),5′-TTCTIAGCGTTT-3′ (SEQ ID NO:23), or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:24).43. The oligonucleotide aptamer of claim 37, wherein the one or moredeoxyinosine nucleotides renders the oligonucleotide aptamer cleavableby an endonuclease V enzymatic activity.
 44. The oligonucleotide aptamerof claim 43, wherein endonuclease V enzymatic activity comprisesThermotoga maritima endonuclease V enzymatic activity.
 45. Theoligonucleotide aptamer of claim 37, complexed with a DNA polymerase.